Abstract
Some methodological controls, believed to be necessary in any study dealing with metabolite (or neurotransmitter) transport by synaptosomes, were performed. The results obtained with [3H]GABA and [14C]glutamic acid are discussed in some detail. The following main conclusions were drawn. The conditions of incubation in which the metabolism of [3H]GABA and [14C]glutamate can be kept within reasonable limits are restricted. For incubation at 37°C and with amino acid concentrations of20 μM or below, the crude mitochondrial fraction (CMF) or synaptosomal protein concentration in the incubation medium should be kept below 0.2–0.4 mg/ml, and the incubation time should not exceed 10 min. The most widely used methods of subcellular particle collection after incubation (Millipore filtration and washing with cold medium or rapid cooling, centrifugation, washing of the pellet and recentrifugation) allow a large ( > 50%) loss of the radioactivity accumulated by the subcellular particles. The results presented suggest that this loss is due to the effect of a sudden lowering of the temperature on the passive permeability of synaptosomal membranes. Alternative methods of particle collection are suggested: (a) a rapid centrifugation procedure by a microcentrifuge; (b) Millipore filtration without filter washing; or (c) Millipore filtration followed by filter washing with warm medium or sucrose. Synaptosomes can be satisfactorily purified from previously incubated CMFs. This permits one to measure the uptake capacity of synaptosomes which suffer no major loss of activity due to the long preparation procedure. When incubated CMFs are pelleted, resuspended in sucrose and subjected to density gradient centrifugation, some leakage of accumulated radioactivity occurs. In spite of this, subcellular particles (synaptosomes or mitochondria) always show a greater uptake of GABA and glutamate when they are isolated from incubated CMFs the when they are first purified and then incubated. GABA/glutamate uptake ratios are different in synaptosomal and mitochondrial fractions, and CMFs show intermediate values of these ratios. The results obtained with CMFs or homogenates cannot be ascribed only to the activity of synaptosomes.
Published Version
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